Repurposing Endonuclease V for the detection and engineering of adenosine-to-inosine editing

项目摘要/摘要 RNA编辑是细胞广泛采用的一种在转录后改变蛋白质序列和基因的策略 表达级别。腺苷-肌苷(A-to-I)编辑是这些RNA最常见和最有影响力的编辑之一 由作用于RNA的腺苷脱氨酶(ADARs)催化。脱氨基改变了 碱基的结构和氢键模式，产生的肌苷与胞嘧啶杂交以 有效地将这些位置重新编码为鸟嘌呤。编辑对于包括胚胎基因在内的许多过程都是必不可少的。 SIS、神经功能和先天细胞免疫，以及功能失调的编辑也与自身免疫有关 疾病、神经紊乱和几种癌症。尽管A-to-I编辑在cell中的关键作用 功能，我们对这种修饰的位置和频率的理解受到固有限制-- 目前可用的转录组中肌苷定位和定量方法中的注释。另外， 选择性地诱导A-to-I编辑的能力对于研究这一修饰将是非常有价值的。 阳离子和新治疗方法的发展，然而目前的方法因其依赖而受到阻碍 关于天然或修饰的ADAR酶的底物结合偏好。一个核心挑战阻碍了 研究A-to-I编辑的方法的发展是缺乏抗肌苷抗体或其他 能够选择性地与这种修饰的核苷酸结合的亲和试剂。我们克服了这一挑战，通过 将自然产生的EndoV蛋白从RNA裂解酶重新定位为RNA结合蛋白 并利用这一点开发了一种工作流程，从细胞总RNA中丰富含肌苷的RNA。我们有 结果表明，这增加了rna-seq数据中含有肌苷的读数的比例，并有助于发现 转录组中新的A-to-I编辑站点。拟议的研究将利用我们的EndoV方法来消除- 开发一个技术工具箱，以推进A-to-I编辑的研究和工程。总之，这些新的 这些方法将使研究人员能够更准确地绘制转录组中的编辑位点，量化 总体编辑流行迅速和高通量，并在活细胞中的特定目标位置直接编辑。 此外，本文所发展的方法也可应用于除ino-2外的其他表位转录修饰。 提供了一套对RNA编辑社区具有广泛实用价值的技术。

Project Summary/Abstract RNA editing is a widespread strategy employed by cells to post-transcriptionally alter protein sequence and gene expression levels. Adenosine-to-inosine (A-to-I) editing is among the most common and impactful of these RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Deamination changes the structure and hydrogen bonding pattern of the nucleobase, and resulting inosines hybridize with cytosine to effectively recode these sites as guanine. Editing is essential for a number of processes including embryogene- sis, neurological function, and innate cellular immunity, and dysfunctional editing is also linked to autoimmune diseases, neurological disorders, and several types of cancer. Despite the critical role of A-to-I editing in cellular function, our understanding of the locations and frequency of this modification are confined by the inherent limi- tations in the currently available methods for mapping and quantifying inosine in the transcriptome. Additionally, the ability to site-selectively induce A-to-I editing would be extremely valuable for both the study of this modifi- cation and the development of new therapeutic approaches, yet current methods are hampered by their reliance on the substrate binding preferences of native or modified ADAR enzymes. A central challenge that has hindered the development of methods for studying A-to-I editing is the lack of availability of anti-inosine antibodies or other affinity reagents capable of selective binding to this modified nucleotide. We have overcome this challenge by repurposing the naturally occurring EndoV protein from an RNA-cleaving enzyme into an RNA-binding protein and have used this to develop a workflow to enrich inosine-containing RNAs from total cellular RNA. We have shown that this increases the fraction of reads in RNA-seq data that contain inosine and facilitates the discovery of new A-to-I editing sites in the transcriptome. The proposed research will leverage our EndoV method to de- velop a toolbox of technologies to advance the study and engineering of A-to-I editing. Together, these new methods will enable researchers to more accurately map editing sites in the transcriptome, quantify changes in overall editing prevalence rapidly and in high-throughput, and direct editing at specific target sites in living cells. Additionally, the methods developed here can be applied to other epitranscriptomic modifications beyond ino- sine, providing a set of technologies that are of broad utility to the RNA editing community.